Plasmin (EC 3.4.21.7, fibrinolysin) is a trypsin-like serine protease which effects protein cleavage at arginine or lysine residues; its principal substrates are fibrin and extracellular matrix (ECM) proteins like fibronectin. Other plasmin substrates include various proteins of the basal membrane, for example, laminin and type IV collagen, and zymogens such as the proforms of urokinase and matrix metalloprotcases. In blood, plasmin is responsible in particular for fibrinolysis, as it cleaves fibrin into soluble fragments. Plasmin is activated by cleavage from its precursor zymogen, plasminogen, by the action of plasminogen activators, principally serine proteases such as urokinase, tPA, and plasma kallikrein (EC 3.4.21.34; kininogenin, PK).
Endogenous plasmin inhibitors such as α2-macroglobulin and α2-antiplasmin, by moderating the anticoagulant effects of plasminogen activators, play key roles in regulating fibrinolysis. Certain pathological conditions (hyperplasminemias) are characterized by dysregulation of plasmin and spontaneous activation of fibrinolysis. The resulting degradation of wound-closing fibrin is exacerbated by the anticoagulant properties of the fibrinogen degradation products, leading to a serious impairment of hemostasis.
Antifibrinolytic drugs are used clinically to treat such conditions; among the commonly used agents are synthetic amino-substituted carboxylic acids such as p-aminomethylbenzoic acid, s-aminocaproic acid, and trans-4-(aminomethyl)-cyclohexanecarboxylic acid (tranexamic acid). These compounds block the binding of plasminogen to fibrin, and thus inhibit the generation of plasmin, but they are not direct inhibitors of plasmin and do not inhibit the activity of already-formed plasmin. A direct antifibrinolytic is aprotinin (TRASYLOL™, Bayer AG, Leverkusen), a 58 amino acid polypeptide obtained from bovine lung. Aprotinin inhibits plasmin with an inhibition constant of 1 nM, but is relatively nonspecific: it effectively inhibits trypsin (Ki=0.1 nM), plasma kallikrein (Ki=30 nM) and, to a lesser extent, a variety of other enzymes.
The principal use of aprotinin was for reduction of blood loss, especially in cardiac surgical procedures with cardiopulmonary bypass (CPB), where it distinctly reduced the need for perioperative blood transfusions (Sodha et al., Expert Rev. Cardiovasc. Ther., 4, 151-160, 2006). Aprotinin was also employed to inhibit blood loss in other operations, for example in organ transplants; it is also used in conjunction with fibrin adhesives.
The use of aprotinin has several disadvantages. Since it is isolated from bovine organs, there is in principle the risk of pathogenic contamination and allergic reactions. The risk of anaphylactic shock is relatively low with the first administration of aprotinin (<0.1%), but increases on repeated administration within 200 days to 4-5%. It has been reported that administration of aprotinin, in direct comparison with ε-aminocaproic acid or tranexamic acid, induces an increased number of side effects (Mangano et al., New Engl. J. Med., 354, 353-365, 2006). Administration of aprotinin led to a doubling of the number of cases of kidney damage requiring dialysis, and the incidence of myocardial infarction and apoplectic stroke was increased in comparison with the control groups. After the Blood Conservation Using Antifibrinolytics in a Randomized Trial (BART) study had shown an increased risk of mortality associated with aprotinin use compared to lysine analogues in high-risk cardiac surgery patients (Fergusson et al., New Engl. J. Med., 358, 2319-2331, 2008), the drug was withdrawn from the market.
A number of synthetic inhibitors of plasmin have been disclosed. Sanders and Seto, J. Med. Chem., 42, 2969-2976, 1999, have described 4-hetero cyclohexanone derivatives with relatively weak activity, with inhibition constants of ≧50 μM for plasmin. Xue and Seto, J. Med. Chem., 48, 6908-6917, 2005, have reported on peptidic cyclohexanone derivatives with IC50 values≧2 μM, but no further development has been reported. Okada (Okada et al., Chem. Pharm. Bull., 48, 1964-1972, 2000; Okada et al., Bioorg. Med. Chem. Lett., 10, 2217-2221, 2000) and Tsuda (Tsuda et al., Chem. Pharm. Bull., 49, 1457-1463, 2001) described derivatives of 4-aminomethyl-cyclohexanoic acid which inhibit plasmin with IC50 values ≧0.1 μM, but clinical use of these inhibitors has not been reported. Potent plasmin inhibitors have recently been described (WO 2008/049595; Dietrich et al., Anesthesiology, 110, 123-130, 2009), but these compounds have limited selectivity and inhibit other trypsin-like serine proteases.
Stürzebecher et al. have described a series of N-terminal sulfonylated benzamidine peptidomimetics having various effects on serine proteases. Included within this class are factor Xa inhibitors, useful as anticoagulants and antithrombotics (U.S. Pat. No. 6,841,701); urokinase inhibitors, useful as tumor suppressors (US Pat. Application Publication No. 2005/0176993, U.S. Pat. No. 6,624,169); inhibitors of plasma kallikrein (PK), factor XIa and factor XIIa, useful as anticoagulants and antithrombotics (US Pat. Application Publication No. 2006/0148901); and matriptase inhibitors, useful as tumor suppressors (US Pat. Application Publication No. 2007/0055065).
Inhibition constants for some compounds affecting plasmin activity have been published in several studies on inhibitors of coagulation proteases. The compounds in question, however, were being investigated as antithrombotics, and therefore a low level of plasmin inhibition was preferred. For example, the thrombin inhibitor melagatran inhibits plasmin with a Ki value of 0.7 μM, and the structurally related compound H317/86 has an inhibition constant of 0.22 μM (Gustafsson et al., Thromb. Haem., 79, 110-118, 1998). However, because both compounds inhibit the protease thrombin much more strongly (Ki≦2 nM), the net effect of administration is inhibition of coagulation. The possibility of using such compounds as pro-coagulants, e.g. for reducing blood loss in cardiac surgical procedures, was not mentioned in any of these papers.
As noted above, aprotinin inhibits not only plasmin but also plasma kallikrein (PK). PK is a multifunctional, trypsin-like serine protease for which several physiological substrates are known. Thus, by proteolytic cleavage, PK is able to release the vasoactive peptide bradykinin from high molecular weight kininogen, and to activate zymogens such as coagulation factor XII, pro-urokinase, plasminogen and pro-MMP 3. It is therefore assumed that the PK/kinin system plays an important role in many pathological conditions, for example in thromboembolic situations, disseminated intravascular coagulation, septic shock, allergies, the postgastrectomy syndrome, arthritis and ARDS (adult respiratory distress syndrome) (Tada et al., Biol. Pharm. Bull, 24, 520-524, 2001).
Accordingly, aprotinin, via its inhibitory effect on PK, inhibits the release of the peptide hormone bradykinin, which in turn has various effects via activation of the bradykinin B2 receptor. The bradykinin-induced release of tPA, NO and prostacyclin from endothelial cells (Schmaier, J. Clin. Invest., 109, 1007-1009, 2002) influences fibrinolysis, blood pressure and inflammatory events. It has been suggested that systemic inflammatory processes which may occur as a side effect in surgical operations can be reduced by inhibiting bradykinin release.
Various bisbenzamidines, such as pentamidine and related compounds, and esters of ω-amino- and ω-guanidinoalkylcarboxylic acids, have been described as PK inhibitors with micromolar Ki values (Asghar et al., Biochim Biophys Acta, 438, 250-264, 1976; Muramatu and Fuji, Biochim. Biophys. Acta, 242, 203-208, 1971; Muramatu and Fuji, Biochim. Biophys. Acta, 268, 221-224, 1972; Ohno et al., Thromb. Res., 19, 579-588, 1980; Muramatu et al., Hoppe-Seyler's Z. Physiol. Chem., 363, 203-211, 1982; Satoh et al., Chem. Pharm. Bull., 33, 647-654, 1985; Teno et al., Chem. Pharm. Bull., 39, 2930-2936, 1991).
The first selective competitive PK inhibitors to be reported (Okamoto et al., Thromb. Res., Suppl. VIII, 131-141, 1988) were derived from arginine or phenylalanine, and inhibit PK with Ki values around 1 μM. Several papers on the development of competitive PK inhibitors have been published by the Okada group, with the most active compounds, derived from trans-4-aminomethylcyclohexanecarbonyl-Phe-4-carboxymethylanilide, having inhibition constants around 0.5 μM (Okada et al., Biopolymers, 51, 41-50, 1999; Okada et al., 2000, Tsuda et al., 2001). It is characteristic of these PK inhibitors that they have a relatively high Ki value.
Potent 4 amidinoaniline PK inhibitors, with Ki values around 1 nM, were described in WO 00/41531, but further development of these compounds was not reported.
Garrett et al. have described transition state analogue PK inhibitors (Garrett et al., J. Pept. Res. 52, 60-71, 1998, Garrett et al., Bioorg. Med. Chem. Lett. 9, 301-306, 1999), but these compounds are prone to non-specific reaction with nucleophiles.
Aliagas-Martin et al., in U.S. Pat. No. 6,472,393, described a wide variety of 4-amidinoanilides which are potent PK inhibitors, having inhibition constants around 1 nM. Antonsson et al. likewise described a wide range of amidine and guanidine PK inhibitors in U.S. Pat. No. 5,602,253. Stürzebecher et al. have described 4-amidino- and 4-guanidino-benzylamines as PK inhibitors, some of which are Factor Xa inhibitors (US Pat. Application Publication. No. 2005/0119190), some of which have a slight inhibitory effect on plasmin (US Pat. Application Publication. No. 2006/0148901), and some of which are dual plasmin/PK inhibitors (PCT Publication No. 2008/049595).
Dyax Corp. has developed a selective plasma kallikrein inhibitor, DX-88 (ecallantide, Kalbitor™), for the treatment of acute attacks in hereditary angioedema. Ecallantide is a recombinant small protein that has been identified utilizing a phage display technology based on the first Kunitz domain of human tissue factor pathway inhibitor (TFPI). Ecallantide is also undergoing phase II clinical testing for the reduction of blood loss during on-pump cardiothoracic surgery (Lehmann, Expert Opin. Biol. Ther., 8, 1187-1199, 2008).
Plasmin and plasma kallikrein, together with approximately 70 other enzymes, belong to the family of trypsin-like serine proteases which share significant sequence homology. In general, this makes it difficult to develop selective inhibitors for a particular protease based on substrate analogues. However, plasmin is missing several amino acids in a loop around the amino acid at position 99, which limits the size of the S2-pocket in most of the trypsin-like serine proteases (binding pocket terminology of Schechter and Berger, Biochem. Biophys. Res. Comm. 27, 157-162, 1967). This leads to a relatively open S2-pocket in the active center of plasmin, which may explain why plasmin has a very broad substrate specificity. Plasma kallikrein (PK) features a glycine at position 99, and the absence of a side chain means that plasma kallikrein also has a relatively open S2 pocket. Based on the X-ray structures of trypsin-like serine proteases in complex with substrate analogue inhibitors (Schweinitz et al., Med. Chem. 2, 349-361, 2006) it appears that the side chains of a P2 L-amino acid and a P3 D-amino acid (side-chain terminology of Schechter and Berger, Biochem. Biophys. Res. Comm. 27, 157-162, 1967) should both be directed towards the enzyme surface.
There remains a need for low-molecular-weight substances, suitable for therapeutic applications, which reversibly and competitively inhibit plasmin, and preferably plasmin and plasma kallikrein together, with high activity and specificity. The present inventors have discovered that it is possible to obtain potent inhibitors of plasmin by a suitable cyclization between the side chains of the P3- and P2-amino acids in substrate analogue inhibitors. Some of these compounds potently inhibit plasma kallikrein as well.
The compounds of the present invention, accordingly, are suitable for modulating and/or maintaining hemostasis in various situations, particularly during and after surgeries with cardiopulmonary bypass, organ transplants, and other major surgical interventions. It is expected that the compounds of the present invention, as inhibitors of plasma kallikrcin, will also lower kinin release, thereby suppressing both kinin-mediated inflammatory reactions and kinin-induced release of tPA from endothelial cells. The latter effect provides an additional mechanism for downregulation of fibrinolysis.